DNA Polymerase I

spinqualitylabelsall models Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Overview DNA replication is catalyzed by DNA polymerase. All cells express several different DNA polymerases that variously participate in the several aspects of DNA replication and in the repair of damaged DNA. DNA polymerases catalyze the reaction (DNA)n residues + dNTP → (DNA)n+1 residues + PPi, where dNTP is the deoxynucleoside triphosphate whose base is complementary to a base on the strand being copied, the so-called template strand. In addition, DNA polymerases cannot initiate replication by linking together two dNTPs, but rather, can only link the incoming nucleotide to a terminal 3'-OH group on an existing polynucleotide strand, the so-called primer strand, thereby forming a 3' → 5' phosphodiester bond between successive deoxynucleotides. If DNA polymerase can only add nucleotides to a pre-existing primer strand, how can the primer be synthesized? The answer is that the initial primer is a short RNA strand that is complementary to the a portion of the template strand and which is synthesized by an RNA polymerase known as primase. This enzyme catalyzes a reaction similar to that catalyzed by DNA polymerase but uses NTPs rather than dNTPs. However primase, as can all RNA polymerases, does not require a primer to initiate polynucleotide synthesis; it can do so by linking together two NTPs in a 3' → 5' linkage. The first known DNA polymerase, an E. coli enzyme now known as DNA polymerase I or Pol I, was discovered and characterized in 1957 by Arthur Kornberg (who received the Nobel prize for this work). Pol I has three active sites: 1. A DNA polymerase. 2. A 3' → 5' exonuclease, that hydrolyzes off mispaired nucleotides at the 3' end of the growing polynucleotide [(DNA)n residues + H2O → (DNA)n-1 residues + dNMP] and hence provides Pol I with the ability to proofread and edit its mistakes. 3. A 5' → 3' exonuclease, whose central role is to remove the RNA primers (although it also participates in DNA repair processes), which the polymerase function then replaces with DNA. These active sites occupy different regions of Pol I. In fact, mild treatment of Pol I by proteases such as trypsin and subtilisin, cleaves Pol I into two catalytically active fragments. The N-terminal fragment (residues 1-323) contains the 5' → 3' exonuclease function, whereas the larger, C-terminal fragment (residues 324-928), which is known as the Klenow fragment, contains both the polymerase and the 3' → 5' exonuclease functions. Structure of the Klenow fragment Thomas Steitz determined the X-ray structure of Klenow fragment in complex with a 13-nucleotide (nt) primer strand and a 10-nt template strand (the primer strand is the strand that is synthesized by the polymerase as the complement of the template strand; the entire DNA is often referred to as primer−template DNA). Here Klenow fragment is shown in ribbon form colored in rainbow order from its N-terminus (blue) to its C-terminus (red). The DNA is drawn in stick form and colored according to atom type with template C cyan, primer C magenta, N blue, O red, and P orange and with an orange rod connecting successive P atoms in each strand. The 3' → 5' exonuclease active site at the N-terminal end of the protein is marked by a Zn2+ ion (gray sphere). 'The arrangement of the polymerase's three domains is reminiscent of a right hand grasping a rod (the DNA) and hence, from N- to C-terminus, they are named “palm“, "fingers", and "thumb". The polymerase's active site is located in the palm domain near the cleft between the fingers and thumb domains. All DNA polymerases of known structure have a similar spatial arrangements of fingers, thumb, and palm domains, even though, in many cases, they have no recognizable sequence similarity with Pol I and the structure of their fingers, thumb, and palm domains bear no resemblance to those of Pol I. The X-ray structure is that of an editing complex, that is, the 3' end of the primer strand, the end that is elongated by the polymerase, occupies the 3'→5' exonuclease active site. This is more clearly seen in a closeup of the DNA in which the the rods connecting successive P atoms have been removed for clarity. Note that the base pair closest to the polymerase active site, a G·C, has opened up to enable the 3' end of the primer strand to reach the exonuclease active site. Click here to hide the protein. Structure of the whole Thermus aquaticus DNA polymerase I As mentioned above, Pol I's primary and essential function is to excise the RNA primers from newly synthesized Okazaki fragments with its 5' → 3' exonuclease function and replace them with DNA using its polymerase function. This yields a double-stranded DNA (dsDNA) with a single strand nick between successive Okazaki fragments, a nick that is eventually sealed through the action of DNA ligase. Pol I from the thermophilic bacterium Thermus aquaticus (Taq, PDB entry 1taq) is 51% identical in sequence with E. coli Pol I, although it lacks a 3' → 5' exonuclease function due to the absence of critical residues. The X-ray structure of the complete Taq Pol I, was also determined by Steitz. Here its C-terminal Klenow fragment portion is initially viewed as is that in the foregoing structure of Klenow·DNA and colored light green, whereas the N-terminal 5' → 3' exonuclease portion is colored in rainbow order from its N-terminus (blue) to its C-terminus (red). Note that there is only tenuous contact between the Klenow fragment and the 5' → 3' exonuclease. Hence, it is unclear how they coordinate their activities to yield a dsDNA molecule with a single nick.

Structures of Klentaq1 in its closed and open forms

Pol I replicates DNA with high fidelity. How does it do so? Gabriel Waksman answered this question by crystallizing the C-terminal domain of Taq polymerase (Klentaq1) with an 11-bp DNA that had a GGAAA-5' overhang at the 5' end of its template strand. The crystals were then soaked in solution containing 2',3'-dideoxy-CTP (ddCTP), which lacks a 3'-OH group, and hence terminates replication after its incorporation at the 3' end of the primer strand. The X-ray structure of these crystals revealed that a ddC residue had been covalently linked to the 3' end of the primer strand, where it formed a Watson–Crick base pair with the 3' G on the template overhang, thus demonstrating the Klentaq1 is enzymatically active in the crystal. In addition, a ddCTP molecule occupied the enzyme's active site, where it formed a Watson–Crick base pair with the template's next G.

spinqualitylabelsall models Klentaq1–Closed conformation (3ktq) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D

spinqualitylabelsall models Klentaq1–Open conformation (2ktq) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D

Here, Klentaq1's N-terminal, palm, fingers and thumb domains are yellow, magenta, green, and blue, respectively. The DNA is drawn in stick form colored according to atom type (template C cyan, primer C green, N blue, O red, and P orange).

In the structure on the left, the crystal had been soaked in a solution of dideoxy-CTP (ddCTP), which the enzyme had added to the 3' end of the primer chain (shown in space-filling form with C green), where it forms a base pair with the a template G. This terminates further primer extension due to the absence of a 3'-OH group at the 3' end of the primer strand. Nevertheless, a ddCTP (shown in space-filling form with C yellow) binds to the enzyme active site at the 3' end of the primer in a base pair with a template G as if it were preparing to add to the 3' end of the primer. In the structure on the right, the ddCTP in the enzyme's active site had been depleted by soaking the crystal in a ddCTP-frree solution. Comparison of these two structures reveals that the structure on the left, the so-called closed conformation, differs from the that on the right, the so-called open conformation, by a hinge-like motion of the fingers domain away from the polymerase active site. The rest of the protein remains very nearly unchanged. This is more readily seen in the morph between the closed and open structures (left, in which, for technical reasons, the ddCTP in the closed conformation is not shown).

This, together with other experimental measurements, indicates that Klentaq1 rapidly samples the available dNTPs in its open conformation, but only when it binds the correct dNTP in a Watson–Crick pairing with the template base does it form the catalytically competent closed conformation. In addition, note how the template G that base pairs with the ddCTP in the closed conformation, moves away from the active site in the open conformation, in which it has no base pairing partner.

A closeup of the active site region in the open conformation (right) reveals that the side chain of the conserved Tyr 671 (colored with C pink) is stacked on top of the template G that forms a base pair with the bound ddCTP, where it apparently participates in verifying that a Watson–Crick base pair has formed. In the closed conformation (left), Tyr 671, which is part of the fingers domain, has moved aside, presumably to permit the active site to form about the incoming dNTP (satisfy yourself that the Tyr 671 side chain is stacked on the template G in the open form but not in the closed form).

3D structures of DNA polymerase

1taq

Additional Resources

For additional information, see: DNA Replication, Repair, and Recombination
For additional information, see: Nucleic Acids

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